The goals of this project are to characterize the molecular mechanisms by which cowpox virus (CPXV) and monkeypox virus (MPXV) inhibit T cell stimulation and the impact of these immune evasion mechanisms on viral virulence and the development of anti-viral immune responses. We previously demonstrated that CPXV inhibits MHC-l-mediated antigen presentation thus escaping poxvirus-specific CD8+ T cells. Preliminary data suggest that, in addition, CPXV escapes CD4+ T cell stimulation and inactivates T cells in trans, i.e. without infecting T cells and independent of antigen presentation. The latter mode of T cell inhibition is very similar to our previously reported mechanism by which MPXV inhibits MHC-independent T cell stimulation. We therefore hypothesize that the responsible gene products are conserved between CPXV and MPXV. By analyzing deletion mutants we identified a gene region in the CPXV genome encoding the genes responsible for both CD4+ T cell escape and inhibiting T cell stimulation by anti-CD3. Since both antigen-dependent-and antigen-independent stimulation of T cells requires signal-transduction by the T cell receptor (TCR), it is conceivable that a single gene is responsible for both T cell evasion phenotypes. In three specific aims, we will (I) identify the genes in CPXV and MPXV responsible for inhibiting MHC-l-independent T cell stimulation by CPXV and MPXV, (II) characterize the corresponding gene products of CPXV and MPXV and determine how they inhibit TCR-mediated T cell activation, and (111) examine the role of T cell evasion for CPXV virulence and the impact of these inhibitory mechanisms on the development of antiviral immune responses in vivo: Since T cell inactivation by CPXV and MPXV is unlike any other viral T cell inhibitor mechanism described to date, our studies are expected to uncover novel molecular strategies for viral immune evasion. We further expect that this work will lead to a re-evaluation of the role of T cell evasion in the pathogenesis of zoonotic orthopoxviruses (and potentially smallpox, if the T cell inhibitors are conserved in Variola major) because these evasion mechanisms are absent from the widely studied Vaccinia Virus (W) and the mousepox virus ectromelia (ECTV).